Project Summary Chronic kidney disease (CKD) is estimated to affect over 14% of US adults and is increasing in prevalence worldwide. The majority of cases are caused by glomerular diseases with sclerotic lesions, and transforming growth factor ? expression in podocytes is a common stress response signal associated with segmental sclerosis. In the previous funding cycle we established a new hypothesis for ?glomerular cell-cell crosstalk? using an inducible transgenic mouse model (PodTbrI) that enables podocytes specific and ligand-independent expression of transforming growth factor ? type I receptor (TbrI) kinase. In this model, TbrI signaling in podocytes results in the release of Edn1, followed by increased Ednra-mediated mitochondrial oxidative stress and dysfunction of adjacent glomerular endothelial cells (GEC), which, in response, release factor(s) that mediate damage and depletion of adjacent podocytes. This was also demonstrated in other models of experimental podocytopathies (Balb/c + Adryamicin, PodDicerKO mice). We identified that a similar stressed endothelial-to-podocyte crosstalk underlies segmental lesions in DKD. We characterized podocyte mitochondrial dynamics in response to transforming growth factor ? signaling. We also performed transcriptomic analysis of isolated GECs after TbrI signaling activation and with this strategy we identified novel GEC injury response pathways. Further, using state of the art proteomics, we identified a panel of novel proteins released by stressed GECs that induce podocyte injury. We also identified key phenotypic markers of cell crosstalk in vivo (GEC ultrastructural changes and loss of glomerular endothelial surface layer). Our findings provide new insights into crosstalk of stressed GECs and podocytes in the pathogenesis of CKD. We hypothesize that the identified GEC secreted proteins mediate podocyte injury and loss in CKD. In this competitive renewal application, we aim to examine and validate the activity of the identified GEC-secreted proteins and cell-cell crosstalk mechanisms mediating podocyte injury in CKD in the following 3 specific aims: SPECIFIC AIMS: 1) To characterize GEC stress and the mechanisms leading to dysfunction and release of crosstalk factors that impact podocytes. 2) To determine GEC response to podocyte activation in vivo. 3) To characterize and validate podocyte responses to GEC secreted factors and determine their functional effects in vitro with a novel 3-D kidney-on-a-chip microfluidic culture system and in vivo. LONG-TERM: The studies proposed in this application will advance our understanding of communications between cells in the glomerulus that underlie the initiation and progression of glomerular disease. The significance of the proposed studies is in the discovery of the requirements and mechanisms for interdependent crosstalk between activated podocytes and stressed endocapillary cells that determine irreversible segmental sclerosis and disease progression. The outcomes will help identify novel diagnostic approaches and potential therapeutic targets for the treatment of glomerular diseases.